Share this article on:

Occurrence of etravirine/rilpivirine-specific resistance mutations selected by efavirenz and nevirapine in Kenyan patients with non-B HIV-1 subtypes failing antiretroviral therapy

Crawford, Keith W.a,b; Njeru, Dorothyc; Maswai, Jonahd; Omondi, Miltond; Apollo, Duncanc; Kimetto, Janed; Gitonga, Lawrencec; Munyao, Jamesd; Langat, Raphaeld; Aoko, Appoloniad; Tarus, Jemutaid; Khamadi, Samoele; Hamm, Tiffany E.a,b

doi: 10.1097/QAD.0000000000000140
Research Letters

Resistance to efavirenz and nevirapine has not been associated with mutations at position 138 of reverse transcriptase. In an evaluation of virologic suppression rates in PEPFAR (President's Emergency Plan For AIDS Relief) clinics in Kenya among patients on first-line therapy (RV288), 63% (617/975) of randomly selected patients on antiretroviral therapy were suppressed (HIV RNA<400 copies/ml). Among those with non-nucleoside reverse transcriptase inhibitor resistance (n = 101), 14 (13.8%) had substitutions at 138 (A, G, K or Q), mutations selected only by etravirine and rilpivirine in subtype B viruses. All 14 patients received efavirenz or nevirapine, not etravirine or rilpivirine, and were predominantly subtype A1. This may be the first report of efavirenz and nevirapine selecting these mutations in these subtypes.

aU.S. Military HIV Research Program, Global Health Programs, Walter Reed Army Institute of Research, Silver Spring

bHenry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, Maryland, USA

cKenya Defense Forces (KDF), United States Army Medical Research Unit – Kenya (USAMRU-K), Nairobi, Kenya

dKenya Medical Research Institute (KEMRI)/Walter Reed Program, South Rift Valley (SRV), US Army Medical Research Unit-Kenya (USAMRU-K), Kerich, Kenya

eSouthern Highlands Walter Reed Program, Mbeya, Tanzania.

Correspondence to Keith Crawford, PhD, RPh, US Military HIV Research Program/Walter Reed Army Institute of Research, Bethesda, Maryland, USA.

Received 4 September, 2013

Revised 5 November, 2013

Accepted 5 November, 2013

HIV-1 group M viruses are responsible for the majority of HIV infections worldwide and are genetically classified into nine subtypes [1]. These subtypes display biological differences in pathogenicity and disease progression [2–7], transmissibility [8–10] and treatment outcomes [11–13]. All currently used antiretroviral drugs were developed, evaluated and approved based on their activity and efficacy for HIV-1 subtype B infections, the predominant subtype in the Western Hemisphere and Europe. However, infections by this subtype are a substantial minority of the HIV-1 infections globally. Antiretroviral drug resistance mutation patterns and resistance algorithms are derived from in-vitro and clinical studies of subtype B viruses and infections. The ‘IAS Annual Resistance Mutation Update’ [14] constitutes resistance data from subtype B viruses, whereas resistance mutation data in the ‘HIV Drug Resistance database’ (HIVdb, Stanford University; are inclusive of mutations from various HIV subtypes. We report here on non-nucleoside reverse transcriptase inhibitor (NNRTI) drug resistance mutations unique to etravirine and rilpilvirine exposure in subtype B infection occurring in patients with non-B subtypes exposed to nevirapine (NPV) or efavirenz (EFV).

RV288 is a multicenter PEPFAR (President's Emergency Plan For AIDS Relief) Basic Program Evaluation that is being conducted in Kenya, Tanzania, Uganda and Nigeria. This cross-sectional study was developed to estimate the rate of virologic suppression of patients on first-line highly active antiretroviral therapy (HAART) in PEPFAR clinics and to identify predictors of virologic suppression and immunologic recovery. Routine virologic monitoring is not a standard of care in any of these counties. Patients on HAART for at least 6 months were randomly selected for a one-time visit which included viral load (HIV RNA) clinical chemistries and hematology, absolute CD4+ lymphocyte count, and percentage and survey of demographics and behavioral risk factors for transmission. Individuals with an HIV RNA above 1000 copies/ml were referred for intensive adherence counseling while having the sample submitted for automated sequencing to detect antiretroviral drug resistance mutations. HIV RNA was quantified from plasma using Roche Amplicor Version 1.5 with lower detection limit of 400 copies/ml in plasma. Specimens with HIV RNA below 400 copies were rerun on a more sensitive platform (Abbott M2000SP/RT). Genotyping was done on archived plasma samples using the Truegene HIV-1 genotyping kit. The study was approved by the Walter Reed Army Institute of Research Institutional Review Board (IRB) and the Kenya Medical Research Institute IRB.

A total of 975 patients were enrolled comprising individuals from Defense Forces Memorial Hospital (Nairobi, n = 325), Kericho District Hospital (Kericho, n = 136), Kombewa District Hospital (Kombewa, n = 109), Nandi Hills District Hospital (Nandi Hills, n = 97), Kapsabet District Hospital (Kapsabet, n = 111), Kapkatet District Hospital (Kapkatet, n = 110) and Litein Mission Hospital (Litein, n = 87). We found 617 out of 975 patients (63%) had HIV RNA below 400 copies, whereas 215 patients (22%) had HIV RNA above 1000 copies/ml, and samples were submitted for resistance testing. Of the 215 samples submitted for resistance testing, 115 (53%) had one or more resistance mutations. Interestingly, 14 of these patients (13.8%) had an amino acid substitution at reverse transcriptase position 138 (A, G, K or Q; Table 1). These mutations are selected by etravirine and rilpivirine in subtype B viruses, resulting in a reduced susceptibility to these drugs, yet no patient had ever been exposed to either of them. All patients were either on EFV or NVP, which are not reported to produce mutations at this position, at least for subtype B viruses. These 14 reverse transcriptase sequences were analyzed for subtype using the Recombinant Identification Program (RIP) tool ( The sequences were submitted to Genbank and their accession numbers are included in Table 1. It appears as if EFV and NVP can select for mutations at position 138 in non-B subtypes, but less so in subtype B viruses (there is one case with subtype B HIV-1).

Table 1

Table 1

Mutations resulting from antiretroviral drug exposure are reported to occur with differing frequencies and display different patterns when subtype B viruses are compared to non-subtype B in the reverse transcriptase, protease and integrase sequences [15–18] Cornelissen et al. [19] identified natural polymorphisms in non-B subtypes that, although not likely to confer significant resistance alone, could modify the effect of drug-selected mutations influencing the fold-sensitivity phenotype. Non-subtype B viruses have acquired resistance to drugs via mutations that are not selected by those same drugs in subtype B viruses in the HIV-1 protease enzyme [20,21]. A particular mutation or genotypic pattern may display differential drug susceptibility across different subtypes [15,22].

Our findings are unique in that they show NNRTI's EFV and NVP producing a cluster of mutations at position 138 in the reverse transcriptase of subtype A, D and recombinants, which are rarely, if ever, observed in subtype B viruses. The mutations produced characteristically result from exposure to etravirine or rilpivirine, to which none of these patients has ever been exposed. In the Stanford database, under a section designated ‘Mutation prevalence according to subtype and treatment’, the E138A mutation has been observed in patients with subtype A and D infections on EFV or NVP, but not E138G, E138K or E138Q for these subtypes. Additionally, the reported frequency of these mutations is quite a bit lower than what we observe in our sample of patients with subtype A virus [23].

Patients failing EFV with just K103N mutation, which usually appears first, are likely to achieve complete suppression with etravirine or rilpivirine, allowing the sequencing of agents within the NNRTI class. Etravirine maintains partial activity even in the presence of certain NNRTI mutations commonly produced by NVP and EFV. The potential for patients with non-B infections to acquire E138 mutations from EFV or NVP increases the chances of patients failing these drugs to have cross-resistance with etravirine and rilpivirine, which can occur by other mutations as well (e.g. Y181C,I; M230L) [24], and emphasizes the importance of resistance testing. Further research is needed to identify factors that may predispose patients on EFV/NVP to acquiring this cluster of mutations.

Back to Top | Article Outline


Conflicts of interest

This research has been supported by the President's Emergency Plan for AIDS Relief (PEPFAR) through the US Department of Defense under the terms of cooperative agreement W81XWH-11-2-0174 with the Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. The views expressed are those of the authors and should not be construed to represent the positions of the US Army or DoD.

Back to Top | Article Outline


1. Gao F, Robertson DL, Carruthers CD, Morrison SG, Jian B, Chen Y, et al. A comprehensive panel of near-full-length clones and reference sequences for nonsubtype B isolates of human immunodeficiency virus type 1. J Virol 1998; 72:5680–5698.
2. Kanki PJ, Hamel DJ, Sankalé JL, Hsieh Cc, Thior I, Barin F, et al. Human immunodeficiency virus type 1 subtypes differ in disease progression. J Infect Dis 1999; 179:68–73.
3. Ssemwanga D, Nsubuga RN, Mayanja BN, Lyagoba F, Magambo B, Yirrell D, et al. Effect of HIV-1 subtypes on disease progression in rural Uganda: a prospective clinical cohort study. PLoS One 2013; 8.
4. Palm AA, Esbjörnsson J, Månsson F, Kvist A, Isberg PE, Biague A, et al. Faster progression to AIDS and AIDS-related death among seroincident individuals infected with recombinant HIV-1 A3/CRF02_AG compared to sub subtype A3. J Infect Dis 2013; doi: 10.1093/infdis/jit416
5. Tarosso L, Sanabani S, Sauer MM, Ribeiro SP, Tomiyama HI, Sucupira MC, et al. HIV-1 subtype BF leads to faster CD4+ T cell loss, compared to subtype B. AIDS Res Hum Retroviruses 2013.
6. Tscherning C, Alaeus A, Fredriksson R, Björndal A, Deng H, Littman DR, et al. Differences in chemokine coreceptor usage between genetic subtypes of HIV-1. J Virology 1998; 241:181–188.
7. Huang W, Eshleman SH, Toma J. Coreceptor tropism in human immunodeficiency virus type 1 subtype D: high prevalence of CXCR4 tropism and heterogeneous composition of viral populations. J Virol 2007; 81:7885–7893.
8. Yang C, Li M, Newman RD, Shi YP, Ayisi J, van Eijk AM, et al. Genetic diversity of HIV-1 in western Kenya: subtype-specific differences in mother-to-child transmission. AIDS 2003; 17:1667–1674.
9. Blackard JT, Renjifo B, Fawzi W, Hertzmark E, Msamanga G, Mwakagile D, et al. HIV-1 LTR subtype and perinatal transmission. Virology 2001; 287:261–265.
10. Renjifo B, Fawzi W, Mwakagile D, Hunter D, Msamanga G, Spiegelman D, et al. Differences in perinatal transmission among human immunodeficiency virus type 1 genotypes. J Hum Virol 2001; 4:16–25.
11. Oyomopito RA, Li PC, Sungkanuparph S, Phanuphak P, Tee KK, Sirisanthana T, et al. Evaluating immunologic response and clinical deterioration in treatment-naive patients initiating first-line therapies infected with HIV-1 CRF01_AE and subtype B. TREAT Asia Studies to Evaluate Resistance (TASER) and The TREAT Asia HIV Observational Database. J Acquir Immune Defic Syndr 2013; 62:293–300.
12. Kantor R, Katzenstein D. Polymorphism in HIV-1 nonsubtype B protease and reverse transcriptase and its potential impact on drug susceptibility and drug resistance evolution. AIDS Rev 2003; 5:25–35.
13. Kyeyune F, Nankya I, Metha S, Akao J, Ndashimye E, Tebit DM, et al. Treatment failure and drug resistance is more frequent in HIV-1 subtype D versus A infected Ugandans: a ten year history in Kampala, Uganda. AIDS 2013; 27:1899–1909.
14. Johnson VA, Calvez V, Gunthard HF, Paredes R, Pillay D, Shafer RW, et al. Update of the drug resistance mutations in HIV-1: March 2013. Top Antivir Med 2013; 21:6–14.
15. Bar-Magen T, Donahue DA, McDonough EI, Kuhl BD, Faltenbacher VH, Xu H, et al. HIV-1 subtype B and C integrase enzymes exhibit differential patterns of resistance to integrase inhibitors in biochemical assays. AIDS 2010; 24:2171–2179.
16. Turner D, Brenner B, Moisi D, Detorio M, Cesaire R, Kurimura T, et al. Nucleotide and amino acid polymorphisms at drug resistance sites in non-B-subtype variants of human immunodeficiency virus type 1. Antimicorb Agents Chemo 2004; 48:2993–2998.
17. Kantor R, Zuenah LS, Shafer RE, Mutetwa S, Johnston E, Lloyd R, et al. HIV-1 subtype C reverse transcriptase and protease genotypes in Zimbabwean patient failing antiretroviral therapy. AIDS Res Hum Retroviruses 2002; 18:1407–1413.
18. Yebra G, de Mulder M, del Romero J, Rodríguez C, Holguín A. HIV-1 non-B subtypes: high transmitted NNRTI-resistance in Spain and impaired genotypic resistance interpretation due to variability. Antiviral Res 2010; 85:409–417.
19. Cornelissen MC, van den Borg R, Zorgdagger F, Lukashov V, Goudsmit J. pol Gene diversity of five human immunodeficiency virus type 1 subtypes: evidence for naturally occurring mutations that contribute to drug resistance, limited recombination patterns, and common ancestry for subtypes B and D. J Virol 1997; 71:6348–6358.
20. Abecasis AB, Deforche K, Snoeck J, Bacheler LT, McKenna P, Carvalho AP, et al. Protease mutation M89I/V is linked to therapy failure in patients infected with the HIV-1 non-B subtypes C, F or G. AIDS 2005; 19:1799–1806.
21. Santos AF, Abecasis AB, Anne-Mieke V, et al. Discordant genotype interpretation and phenotypic role of protease mutations in HIV-1 subtypes B and G. J Antimicrob Agents Chemo 2009; 63:593–599.
22. Snoeck J, Kantor R, Shafer R, Van Laethem K, Deforche K, Carvalho AP, et al. Discordances between interpretation algorithms for genotypic resistance to protease inhibitors of human immunodeficiency virus are subtype dependent. Antimicrob Agents Chemo 2006; 50:694–701.
23. Rhee SY, Kantor R, Katzenstein DA, Camacho R, Morris L, Sirivichayakul S, et al. for the international Non Subtype B HIV-1 Working GroupHIV-1 pol mutation frequency by subtype and treatment experience: extension of the HIVseq program to seven non-B subtypes. AIDS 2006; 20:643–651.
24. Vergne L, Snoeck J, Aghokeng A. Genotypic drug resistance interpretation algorithms display high levels of discordance when applied to non-B strains from HIV-1 naive and treated patients. FEMS Immunol Med Microbiol 2006; 46:53–62.
© 2014 Lippincott Williams & Wilkins, Inc.